Differential expression profile of plasma exosomal microRNAs in acute type A aortic dissection with acute lung injury

MicroRNAs (miRNAs) packaged into exosomes mediate cell communication and contribute to the pathogenesis of acute type A aortic dissection (ATAAD) with acute lung injury (ALI). The expression profile of plasma exosomal miRNAs in ATAAD patients with ALI hasn’t been identified. We performed a miRNA-sequencing to analyze the differentially expressed miRNAs (DE-miRNAs) of circulating exosomes in ATAAD patients with ALI compared to patients without ALI, founding 283 specific miRNAs in two groups. We respectively selected the top 10 downregulated and upregulated DE-miRNAs for further studies. The predicted transcription factors (TFs) of these DE-miRNAs were SMAD2, SRSF1, USF1, etc. The Gene Ontology (GO) and Kyoto Encyclopedia Genes and Genomes (KEGG) analysis predicted their target genes mainly involved acute inflammatory response, cell junction, cytoskeleton, NF-κB signaling pathway, etc. Construction and analysis of the PPI network revealed that RHOA and INSR were considered hub genes with the highest connectivity degrees. Moreover, we confirmed two exosomal miRNAs (hsa-miR-485-5p and hsa-miR-206) by real-time quantitative polymerase chain reaction (RT-qPCR) in a validation cohort. Our study identified a plasma exosomal miRNAs signature related to ATAAD with ALI. Certain DE-miRNAs may contribute to the progression of this disease, which help us better understand the pathogenesis of ATAAD with ALI.


Prediction of transcription factors (TFs) of differentially expressed miRNAs (DE-miR-NAs).
Based on the sequencing results above, we concentrated on the following functional study of the top 10 candidates down-and upregulated miRNAs. Their upstream TFs were predicted by FunRich software. We listed the top 10 TFs for these DE-miRNAs, which were shown in Fig. 3a www.nature.com/scientificreports/ Gene Ontology (GO) functional and Kyoto Encyclopedia Genes and Genomes (KEGG) pathway analysis. Initially, we used the TargetScan, miRbase and RNAhybrid websites to predict the target genes of the top 10 DE-miRNAs. Additionally, to better understand the biological function and pathways of the predictive target genes, we performed GO and KEGG pathway analysis. The biological process (BP), cellular component (CC) and molecular functions (MF) of GO analysis were summarized in Fig. 4a-c. The changes of BP were enriched in the response to hypoxia, natural killer (NK) cell proliferation, NK T cell proliferation, acute inflammatory response, etc. (as shown in Fig. 4a), and the changes of CC were enriched in the cell junction, cytoskeleton, etc. (as shown in Fig. 4b). Figure 4c suggested that the changes of MF were manifested in the protein binding, complement component C4b and C3b receptor activity, sulfite oxidase activity, etc. The KEGG pathway analysis was presented in Fig. 4d, and these predicted target genes were primarily involved in adhesion junction, necroptosis, NF-κB signaling pathway, etc.
Protein-protein interaction (PPI) network construction. We submitted those predictive target genes of the candidate DE-miRNAs to the STRING database and constructed PPI networks for them, which was shown in Fig. 5a,b. Using a comprehensive score greater than 0.4, we obtained 55 and 125 node pairs for target genes of downregulated and upregulated DE-miRNAs, respectively. Then, to identify the hub genes of the two PPI networks, we input these node pairs into Cytoscape, and the top 10 hub genes calculated by degree scoring were presented in www.nature.com/scientificreports/

Discussion
In this study, we analyzed the profile of plasma exosomal miRNAs in ATAAD patients with ALI and selected the top 10 downregulated and upregulated DE-miRNAs in 283 specific miRNAs for further functional analysis. Initially, SMAD2, MYOCD, MKL1, SMAD4, SMAD3, NR3C1, GTF2I, NFIC, SRC, and RREB1 were predicted as the TFs of the downregulated DE-miRNAs and CEBPB, IL4, HOXD10, GLI1, SRSF1, ZMYND8, CCNT2, USF1, MKL1 and HMCA1 were predicted as the TFs of the upregulated DE-miRNAs. Additionally, the biological functions and pathways of predictive target genes of these DE-miRNAs were involved in the regulation of response to hypoxia, NK cell proliferation, NK T cell proliferation, acute inflammatory response, cell junction, cytoskeleton, HIF-1 signaling pathway, NF-κB signaling pathway, necroptosis and adherent junction. Finally, RHOA and INSR were considered hub genes in predictive target genes of DE-miRNAs. For further validation, we found that the expression of hsa-miR-485-5p and hsa-miR-206 showed the same trend with regard to sequencing results, which indicated their regulatory role in the pathogenesis of ATAAD with ALI. Preoperative ALI is a frequent and severe complication in ATAAD, and hypoxemia is the most typical symptom in these patients. It has been reported that ATAAD patients complicated with ALI have longer the duration of ventilation and ICU and hospital stays, which leads to a poor clinical prognosis 17 . As a result, a better understanding of the pathophysiology of the ATAAD with ALI can improve clinical prophylaxis and treatments in  www.nature.com/scientificreports/ these patients. Exosomes are small membrane vesicles that are rich in miRNAs and can protect miRNAs from the degradation of RNase in circulation, which act as the carriers of intercellular communication 18 . Recent studies revealed that plasma exosomal miRNAs are closely related to the onset of ALI 19,20 . Jiang et al. 20 explored the influence of circulating exosomes on macrophage activation of sepsis-related ALI and demonstrated that exosomal miR-155 promoted macrophages proliferation and inflammation by targeting SHIP1 and SOCS1. Another study showed that exosome-shuttling miR-1-3p increased PMVECs permeability and membrane injury by targeting SERP1, leading to vascular barrier dysfunction 19 . However, the association between the plasma exosomal miR-NAs and ATAAD with ALI has not been reported. In our present study, we conducted RNA sequencing and identified a total of 283 specific plasma exosomal miRNAs in ATAAD patients with ALI. Based on its pathophysiological process, these miRNAs might be released from a variety of activated inflammatory cells (e.g. neutrophils, lymphocytes, monocytes, etc.), aortic and pulmonary vascular endothelial cells, and even alveolar epithelial cells via exosomes, which were transferred into the circulation 21,22 . In these DE-miRNAs, we eliminated those novel miRNAs or low expression level miRNAs in each sample and finally, selected the top 10 downregulated (hsa-miR-181d-5p, hsa-miR-31-3p, hsa-miR-326, hsa-miR-423-5p, hsa-miR-145-5p, hsa-miR-744-5p, hsa-miR-99b-3p, hsa-miR-214-3p, hsa-miR-206, hsa-let-7e-5p) and upregulated (hsa-miR-103a-3p, hsa-miR-26b-3p, hsa-miR-1306-5p, hsa-miR-7-1-3p, hsa-miR-3120-3p, hsa-miR-335-3p, hsa-miR-485-5p, hsa-miR-664a-3p, hsa-miR-29a-3p, hsa-miR-483-3p) DE-miRNAs for further studies.
By predicting TFs of these DE-miRNAs, we found that SMAD2, MYOCD, MKL1, SMAD4, SMAD3, NR3C1, GTF2I, NFIC, SRC, RREB1 were predicted to be the downregulated DE-miRNAs TF, while CEBPB, IL4, HOXD10, GLI1, SRSF1, ZMYND8, CCNT2, USF1, MKL1 and HMCA1 proteins were predicted to be the upregulated DE-miRNAs TF. Among these TFs, the SMAD proteins family might potentially modulate the expression of the main downregulated DE-miRNAs. Previous studies showed that SMAD proteins could control DROSHA-mediated miRNA maturation in human smooth muscle cells 23 . Recent research expanded the results and indicated that they were involved in miRNA transcription. For instance, Du et al. 24 demonstrated that SMAD4 regulated miR-425-TGF-β signaling pathway, resulting in granulosa cell apoptosis. Their influence on miRNAs in osteoclast differentiation was also well documented 25 . In the predictive TFs of upregulated DE-miRNAs, SRSF1, an RNA binding protein, had been shown to modulate miRNAs processing such as miR-7, miR-10b, miR-29b 26 . Studies indicated that SRSF1 regulated the expression of miR-7 through binding to the pri-miR-7 sequence and promoting DROSHA cleavage 27 . It can also modulate miR-10b and miR-29b in the same way, which played a critical role in immune response and tumorigenesis 28,29 . Besides, USF1 can activate the expression of miR-132 by binding to the E-box in the miR-132 promoter and influence its synthesis in oxygen and glucose deprivation-induced cell apoptosis 30 . More researches about the role of these predictive TFs in ATAAD with ALI need to be further conducted.
According to GO and KEGG functional analysis, the top 20 enrichments in BP, CC and MF of GO were listed and showed that certain miRNAs participate in biological processes or functions associated with response to Figure 6. Validation of candidate DE-miRNAs by RT-qPCR in the Non-ALI and ALI groups. (a-h) Relative expression levels of selected miRNAs in the Non-ALI and ALI groups were detected by RT-qPCR (n = 20 in each group). The results were normalized to cel-miR-39. Data were analyzed by Mann-Whitney U test. Hsa-miR-181d-5p and hsa-miR-26b-3p could not be detected owing to their low expression levels. DE-miRNAs differentially expressed miRNAs; RT-qPCR real-time quantitative polymerase chain reaction; Non-ALI group patients with acute type A aortic dissection without acute lung injury, ALI group patients with acute type A aortic dissection with acute lung injury. *P < 0.05; **P < 0.01. www.nature.com/scientificreports/ hypoxia, natural killer (NK) cell proliferation, NK T cell proliferation, acute inflammatory response, cell junction and cytoskeleton. Numerous evidence has supported that ATAAD with ALI is characterized by widespread uncontrolled inflammation in the lungs, a functional or structural breakdown in pulmonary vascular endothelial junction and barrier damage, resulting in severe oxygenation impairment in body 31 . Hypoxia can be further pro-inflammatory and lead to a breakdown of vascular barriers. But the adaptive response to hypoxia, especially hypoxia-inducible transcription factors (HIF) stabilization and induction of its downstream genes, bears anti-inflammatory and lung tissue-protective aspects, which highlight the functional role of hypoxia signaling in ALI 32 . It has been reported that the expression of many miRNAs changes under hypoxia, and these miRNAs may modulate HIF expression or switch, but the results are controversial 33 . Besides, most of these studies have focused on cancer cell lines only and often ignore endothelial or epithelial cells, so that the exact role of hypoxiainduced miRNAs in them remains unclear 33 . Our results above further, suggested that specific plasma exosomal miRNAs were involved in the pathogenesis of ATAAD with ALI by recruiting immune cells, expanding inflammatory response, destroying cell junction and skeleton (a model figure was shown in Supplementary Fig. S3).

Scientific Reports
In addition, HIF-1 signaling pathway, NF-κB signaling pathway, necroptosis and adherens junction were listed in the top 20 results of KEGG analysis, and these biological pathways were also closely related to ATAAD with ALI 34 . Recent evidence revealed that the activation of the HIF-1 signaling pathway could amplify the inflammatory response by stimulating the release of inflammatory factors, and promote the apoptosis of alveolar type II epithelial (AT-II) cells, which exacerbated the ALI 35 . The NF-κB family controls various processes such as immunity and inflammation 36 . Chen et al. 37 confirmed that NF-κB signaling pathway was abnormally activated in the AT-II of patients with ALI and promoted the production of inflammatory factors including IL-1β, IL-6 and TNF-α, which initiated the inflammatory cascade. Besides, necroptosis might be involved in endothelial injury 38 and adherens junction were critical for basal pulmonary microvascular integrity 39 . These results were similar to those of GO analysis, indicating the potential role of selected DE-miRNAs in ATTAD with ALI. Next, we constructed the PPI network of those predicted target genes and identified the top 10 hub genes. Inspiringly, the changes in the 20 genes expression were generally consistent with previous studies' results. For the upregulated genes, we found that RAS homolog family member A (RHOA) ranked first. Recent studies showed that RHOA and its downstream effector were closely related to the pathogenesis of ALI 40 . Upregulation of the RHOA/Rho kinase (ROCK) signaling pathway promotes immune cell migration and adhesion, accelerates pulmonary endothelial cells' apoptosis, and these effects result in endothelium barrier dysregulation and edema-the hallmarks of ALI 26 . Besides, other hub genes such as janus kinase 3 (JAK3) 41 , calmodulin 1 (CALM1) 42 and ribosomal protein L13 (RPL13) 43 have also been reported to act as risk factors involved in the onset of ALI. For the downregulated genes, insulin receptors (INSR) had the highest connectivity degrees. It binds insulin to produce biological effects and is positively correlated with insulin level in the body 44 . Emerging evidence revealed that hyperinsulinemia had a protective effect on obese rats with lipopolysaccharide (LPS)-induced ALI by increasing their alveolar fluid clearance 45 . Furthermore, peroxisome proliferator-activated receptor-alpha (PPARA), as the predictive downregulated hub gene, has also been reported that it reduces inflammation and vascular leakage in a murine model of ALI 46 . These hub genes mentioned above may play a potential role in exploring novel mechanisms and therapeutic targets in ATAAD with ALI.
Moreover, based on sequencing results and previous bibliography retrieval, we selected 10 candidate miRNAs for validation. The RT-qPCR confirmed that hsa-miR-485-5p was significantly upregulated and hsa-miR-206 was significantly downregulated in ALI group. Of the two specific miRNAs, miR-206 has been previously reported to be associated with ALI. Zhou et al. 47 demonstrated that miR-206 expression was significantly decreased in lung tissue of sepsis-induced ALI mouse model, showing the same trend as our findings in plasma exosomes in ALI group, and the phenotype of ALI including the lung tissue inflammatory response, wet to dry weight ratio (W/D) and bronchoalveolar lavage fluid (BALF) could be attenuated by injection of miR-206 agomir. Further in vitro experiments revealed that downregulated miR-206 increased the permeability of alveolar air-blood barrier and promoted the development of ALI by targeting CX43 mRNA and upregulating CX43 expression in AT-II cells. These pathological changes above are also common in ATAAD-induced ALI 4 , suggesting a potential role of miR-206 in its pathogenesis. What's more, miR-206 was found to be related to inflammation, apoptosis and autophagy 8,48,49 . For miR-485-5p, a relative study with reference to ATAAD or ALI is lacking. Existing evidence showed that it mediated inflammatory factors (TNF-α, IL-6, IL-8, etc.) involved in inflammation 50 . Other studies have reported its association with suppression of cell proliferation and migration 51,52 . Thus, we speculate that miR-485-5p may be involved in ATAAD with ALI by regulating inflammation and promoting pulmonary capillary endothelial damage. However, its precise function in this process remains to be elucidated.
As far as we know, this study is the first to explore the DE-miRNAs in plasma exosomes from ATAAD patients with ALI. We measured the plasma exosomal miRNA profile of these patients, which provided us with a novel source of biomarkers for this disease. In addition, plasma exosomal miRNAs bind and enter their target cells through the circulation to regulate the post-transcriptional process. Such mediation can help us better clarify the underlying mechanisms of ATAAD with ALI. However, there are still several limitations. Firstly, the validation cohort in this study was relatively small. The results need to be verified in a larger population. Secondly, the present study was lack of in vitro and in vivo experimental validation of the functions of the two confirmed exosomal miRNAs in ATAAD with ALI. Corresponding experiments will be performed in the future to gain insight into its pathogenesis.

Conclusion
In summary, this study has identified differential expression profile of plasma exosomal miRNAs in ATAAD with ALI, which were related to inflammation and dysfunction of cells junction. Further research is required to explore their potential role in pathogenic mechanisms.  19 Patients were enrolled as the discovery cohort (Non-ALI group, n = 8; ALI group, n = 11) and 40 patients were enrolled as the validation cohort (Non-ALI group, n = 20; ALI group, n = 20). Plasma exosome miRNAs were evaluated by next-generation sequencing (NGS) in the discovery cohort, and then, selected miRNA candidates from the differential expression profile of plasma exosomal miR-NAs were validated by RT-qPCR in the validation cohort. The diagnosis of ALI was based on the American-European Consensus Conference (AECC) definition 54 . The following four criteria were required to be present: (1) acute onset; (2) P aO2 /F iO2 ≤ 300 mmHg [regardless of positive end-expiratory pressure (PEEP) level]; (3) bilateral infiltrates seen on frontal chest radiograph; (4) pulmonary artery wedge pressure (PAWP) ≤ 18 mmHg when measured or no clinical evidence of left atrial hypertension. Patients with the following conditions were excluded: those with chronic aortic dissection, chronic lung diseases, chronic renal or liver failure, immune system diseases, connective tissue diseases or malignant diseases.
The present study was approved by the Medical Ethical Committee of the Xiangya Hospital of Central South University (Hunan, China) and conducted following their guidelines and regulations. All patients provided written informed consent.
Exosome isolation. Whole blood (5-10 ml) was sampled from each patient on admission, centrifuged at 3000 rpm for 10 min at 4 °C. The collected plasma samples were stored − 80 °C until required. Exosomes were extracted by differential ultra-centrifugation as follows. Briefly, plasma (4 ml) was centrifuged at 12,000 rpm for 10 min at 4 °C to remove cell debris. The supernatant fluid was diluted with 20 ml phosphate-buffered saline (PBS) and filtered through a 0.22 µm disposable filter. Then, it was transferred to a ultracentrifuge (Beckman Coulter, Inc., Brea, CA) and ultracentrifuged using a Type 70Ti rotor (Beckman Coulter, Inc., Brea, CA) at 120,000×g for 90 min at 4 °C twice. The pellets containing exosomes were suspended in 200 µl PBS for further application.
TEM. For electron microscopy analysis, isolated exosomes suspended in PBS were dropped on copper-coated grids and stained with 2% phosphotungstic acid. After the grids were completely dried at room temperature, they were visualized using a Hitachi 7800 TEM (H-7800, Hitachi Ltd., Tokyo, Japan).
NTA. NTA were performed using a NanoFCM N30E instrument (N30E, Fujian, China) according to the manufacturer's instruction. The exosome samples were diluted in PBS. The NanoFCM instrument captured the Brownian motion of the extracted nanoparticles and measured their size and concentration.
Western blot. The isolated exosomes were lysed in RIPA lysis buffer containing a protease inhibitor to extract protein and the samples were quantified by a BCA protein assay kit. Equal amounts of protein were separated on a 10% or 15% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), and the bands were transferred to polyvinylidene fluoride (PVGF) membranes. After blocking with 5% skimmed milk for 1 h, the membranes were incubated primary antibodies at 4 °C overnight and then incubated with peroxidase-conjugated anti-rabbit or anti-mouse secondary antibodies for 1 h at room temperature. Finally, the membranes were visualized and analyzed using the ChemiDoc MP Imaging system (Bio-Rad, Hercules, CA, USA).

Exosomal RNAs extraction and NGS.
Total RNA was extracted from plasma exosomes samples using miRNeasy ® Mini kit (Qiagen, Cat. No. 217004). The RNA quality and quantity were evaluated by the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 System (Agilent Technologies, CA, USA). Then, the samples were used to establish a sequencing library and the quality of the library was tested with an Agilent 2100 bioanalyzer before the NGS.
Small RNA sequencing (16-30nt) was performed using the BGISEQ-500 sequencer (BGI Park, Shenzhen, China). Low-quality data were removed, and a substantial amount of small RNAs, which mapped perfectly to the human genome (ftp:// ncbi. nlm. nih. gov/ genom es/ Homo_ sapie ns) were obtained for further analysis. The miRNA expression levels were estimated by the value of fragments perkiobase million (FPKM) and their relative expression levels were assessed by the DEGseq method. Quantitative data for each miRNA from the ALI and non-ALI group was compared to obtain absolute fold change (FC) values, and then log 2 FC transformation were performed. Differentially expressed miRNAs (DE-miRNAs) was defined by an absolute value of log 2 FC > 0.8, the Q-value (adjusted P-value) < 0.05 and the value of FPKM in each sample > 0.
Prediction of miRNAs potential TFs. The upstream TFs of DE-miRNAs were predicted using FunRich software 55 . The selected up-regulated or down-regulated miRNAs were inputted into the software, and the results showed the top 10 predictive TFs.